Site directed gene mutagenesis kit

In brief, the 3′-UTR of MST4 mRNA containing the miR-593-5p binding site was PCR amplified. Wild-type and mutant MST4 3′-UTRs containing the putative binding region of miR-593-5p were constructed and cloned into the pmiR-REPORT vector (Ambion, Foster City, CA, USA). Site-directed Gene Mutagenesis Kit (Roche, Basle, Switzerland) was used to produce the mutations of the miR-593-5p targeting site. The luciferase activities were measured at 48 hours after cotrans-fection with Dual-Luciferase Reporter Assay system (Pro-mega, Madison, WI, USA). Then, vector containing Renilla luciferase was co-transfected as a reference control. The experiments were performed in triplicate and repeated three times.

Generally, UTR is the 3′-UTR of EIF4A1 mRNA, and has a concentration of miR-1284 with side binding, with PCR amplification. Mutant EIF4A1 3′-UTR and wild-type with putative binding regions of miR-1284 content have been cloned and constructed into the vector of pmiR-REPORT (Ambion). Site-directed Gene Mutagenesis Kit (Roche, Basle, Switzerland) was used in producing the target site in the miR-1284 mutations. After 48 hrs of co-transfection, luciferase activities were studied using the dual luciferase reporter (promega, Madison, WI) assay system. The vector containing Renilla luciferase was used as the reference control for co-transfection. All the experiments were performed in triplicate.